|Year : 2013 | Volume
| Issue : 3 | Page : 210-216
Anatomical and functional changes in the cornea and retina after ultraviolet-A riboflavin cross-linking in patients with keratoconus
Kamal Enam, Eman Azmy, Abd El-monem Abou El-Fetouh
Department of Ophthalmology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
|Date of Submission||15-May-2013|
|Date of Acceptance||10-Aug-2013|
|Date of Web Publication||28-Feb-2014|
Department of Ophthalmology, Faculty of Medicine, Mansoura University, Mansoura
Source of Support: None, Conflict of Interest: None
The aim of this study was to evaluate the efficacy of corneal collagen cross-linking in stabilization of the keratoconus and the safety of the procedure in the cornea and retina.
This study is a prospective interventional case series study.
Patients and methods
The study included 19 eyes of 19 patients with grade II-III keratoconus. All of them were treated with riboflavin ultraviolet-A collagen cross-linking. The patients were followed up monthly for 6 months. Examination of each patient included evaluation of the uncorrected visual acuity, best-corrected visual acuity, and spherical equivalent; endothelial cell counting; anterior segment optical coherence tomography using a corneal Scheimpflug camera; posterior segment assessment by fluorescein angiography; posterior segment optical coherence tomography; and multifocal electroretinography.
Uncorrected visual acuity and best-corrected visual acuity showed statistically significant improvement. The mean improvement in spherical equivalent was 1.5 (1.0) D. The K-value at the apex significantly decreased (the mean decrease was 1.40 D). There was a significant decrease in the corneal pachymetry at the thinnest location at the first month postoperatively; however, at 3 months postoperatively, the corneal pachymetry increased gradually, reaching stability at the end of the follow-up period. There was a significant increase in the mean intraocular pressure of the treated eyes. Of the treated eyes, 13 showed a demarcation line at a depth of 300 10.5 mm. The preoperative mean endothelial cell count showed a statistically significant decrease. Retinal fluorescein angiography, optical coherence tomography, and multifocal electroretinography at the foveal ring showed no abnormalities in the riboflavin-treated eyes.
Corneal collagen cross-linking is an effective procedure in stopping the progress of keratoconus and is safe for both anterior and posterior segments.
Keywords: Keratoconus, multifocal electroretinography, riboflavin ultraviolet-A cross-linking, Scheimpflug camera
|How to cite this article:|
Enam K, Azmy E, El-Fetouh AmA. Anatomical and functional changes in the cornea and retina after ultraviolet-A riboflavin cross-linking in patients with keratoconus. J Egypt Ophthalmol Soc 2013;106:210-6
|How to cite this URL:|
Enam K, Azmy E, El-Fetouh AmA. Anatomical and functional changes in the cornea and retina after ultraviolet-A riboflavin cross-linking in patients with keratoconus. J Egypt Ophthalmol Soc [serial online] 2013 [cited 2019 Aug 19];106:210-6. Available from: http://www.jeos.eg.net/text.asp?2013/106/3/210/127407
| Introduction|| |
Keratoconus is an asymmetric, bilateral, progressive and noninflammatory ectasia due to gradual biomechanical instability of the cornea. Its reported frequency is ∼1 : 2000 in the general population. The condition usually develops at puberty and progresses in ∼20% of patients to such an extent that penetrating keratoplasty becomes necessary to preserve vision .
In most cases, corrective lenses fitted by a specialist are effective enough to allow the patient to continue normal functioning. Further progression of the disease may require surgery, for which several options are available, including implantation with intrastromal corneal ring segments, cross-linking, mini asymmetric radial keratotomy, deep anterior lamellar keratoplasty, Epikeratophakia, and, in 25% of cases, penetrating corneal transplantation ,,,,,.
Corneal collagen cross-linking (CXL) has been introduced to reduce the progression of noninflammatory corneal degeneration such as keratoconus, pellucid marginal degeneration, and corneal ectatic disorder after corneal refractive procedures ,,.
The standard treatment includes CXL using the photosensitizer riboflavin and ultraviolet-A (UV-A) light preceded by corneal epithelial removal; it could be carried out transepithelially without removal of the epithelium ,.
Since the early 1990s, basic laboratory studies have demonstrated that CXL causes a long-term increase in corneal biomechanical rigidity by stiffening the human cornea by more than 300%, increasing the collagen fiber diameter by 12.2%, and leading to the formation of high molecular weight collagen polymers with a remarkable chemical stability. Subsequent clinical studies have shown the safety and efficacy of the procedure in reducing the deterioration associated with normally progressive corneal disorder ,.
| Aim|| |
The aim of this study was to evaluate the efficacy of CXL in stabilization of the keratoconus and the safety of the procedure in the cornea and retina.
| Patients and methods|| |
This prospective interventional case series study included 19 eyes of 19 patients with keratoconus who attended the outpatient clinic of Mansoura Ophthalmic Center from January 2011 to January 2012. The study was approved by the local institutional review board, and the research procedures used in this study followed the tenets of the Declaration of Helsinki. The nature of the procedure was explained to the participants in detail, and a written informed consent form was submitted by all participants before inclusion in the study. The patients included in the study were those with grade II-III keratoconus (according to the Amsler-Krumisch classification),  those with evidence of progressive keratoconus confirmed by an increase in maximal curvature on computerized corneal topography by at least 1.00 D in the previous 6 months, those with a corneal thickness of more than 400 mm, and those between 18 and 60 years of age.
Patients with a corneal thickness of less than 400 mm at the thinnest point; those who were pregnant; those with a history of herpetic keratitis, severe dry eye, and concurrent corneal infections; and those with an endothelial cell count of less than 1000/mm 2 concomitant autoimmune diseases were excluded from the study. The other eye of the included patients was excluded either because it had not met the inclusion criteria or because it was being prepared for an alternative line of management.
Preoperative examinations included evaluation of uncorrected visual acuity (UCVA) and best-corrected visual acuity (BCVA; using glasses or CL) using Snellen charts, which were transformed into the logarithm of the minimum angle of resolution (log MAR) for further statistical analysis as recommended by Holladay , slit-lamp examination, noncycloplegic refraction using a Topcon automated refractometer and/or retinoscopy, intraocular pressure (IOP) measurement using the (Tono-Pen), and fundus examination.
Specific investigations for anterior segment assessment
Anterior segment assessment was carried out using wavelight Oculyzer II Pentacam (Alcon Surgical, USA), which uses the rotating Scheimpflug camera; the rotational measuring procedure generates crisp Scheimpflug images in three dimensions, with the dot matrix fine meshed in the center due to the rotation. It generates a three-dimensional model of the anterior eye segment from as many as 25 000 true elevation points ,. The topography and pachymetry of the entire anterior and posterior surface of the cornea from limbus to limbus were evaluated. Anterior segment optical coherence tomography (OCT) was carried out using Cirrus HD-OCT (model 4000; Carl Zeiss Meditec, Germany); the corneal endothelial cell count was obtained using the noncontact specular microscope (model EM 3000; Tomey, USA).
Specific investigations for posterior segment assessment
Fluorescein angiography was performed using the Topcon TRC 50 IX fundus camera. Leakage was evaluated by comparing the early (taken at 1-2 min) and late frames (taken at 5-6 min). Good quality frames were used without further enhancement.
Foveal central thickness (FCT) was measured for all eyes using Cirrus HD-OCT (model 4000; Carl Zeiss Meditec). It scans a cube of 6.0 × 6.0 mm length with a resolution of 512 × 128, fixating on the macula. The printout includes the FCT and the thickness map, which is a circle with a diameter of 6 mm centered at the foveola. The map divides the macula into nine ETDRS regions.
RETIscan21, version 07/01 (Roland instruments) was used for multifocal electroretinography (mERG). Signals were picked up by electrodes placed at specific regions of the patient's head. The electrodes were active HK-loop electrodes, reference electrodes, and ground EEG electrodes that were made of silver. The first electrode was attached to the lower lid with its thread (loop) touching the globe just below the cornea; the other electrodes were attached to the patient's head (forehead and temple) after cleaning the skin and placing conductive plastic (TEN20). The location of mERG stimuli and anatomic areas corresponded roughly as follows: Ring 1 to the fovea (R1 = central 2°), ring 2 to the parafovea (R1 = central 2-5°), ring 3 to the perifovea (R1 = central 5-10°), ring 4 to the near periphery (R1 = central 10-15°), and ring 5 to the middle periphery (R1 > 15°).
Corneal collagen cross-linking procedure
All patients underwent CXL as a day surgery procedure. The procedure was carried out under sterile conditions in the operating theater. Topical anesthetic eye drops were applied. Pilocarpine (2%) drops were administered in the eye to be treated to reduce the amount of light rays, which are potentially harmful, reaching the lens and retina. Corneal epithelial debridement was carried out in a central 9-mm area using a sponge. Riboflavin (0.1%) solution in 20% dextran (MedioCross; Medio-Haus Medizinproduct Gmbh, Brunswiker, Germany) was applied to the cornea every 3 min for 30 min. The saturation of the cornea with riboflavin and its presence in the anterior chamber was monitored closely by slit-lamp inspection (blue filter) before treatment. Riboflavin saturation ensures the formation of free radicals, whereas riboflavin shielding ensures the protection of deeper the structure, such as the corneal endothelium. The cornea was exposed to UV rays emanating from a solid state device CBM VEGA X-linker (CSO, Florence, Italy). Exposure lasted for 30 min, during which time the riboflavin solution was applied but only every 5 min to saturate the cornea with riboflavin and prevent it from drying.
Postoperatively, patients received cyclopentolate hydrochloride and levofloxacin eye drops. Bandage soft contact lenses were applied until re-epithelialization was complete.
Patients were followed up at 1, 2, 3, 4, 5, and 6 months postoperatively. At each follow-up visit, UCVA, noncycloplegic refraction, BCVA, and the corneal surface (using Pentacam) were assessed and anterior segment OCT and specular microscopy were carried out. The posterior segment was followed up at 1, 3, and 6 months postoperative by fundus examination, fluorescein angiography, posterior segment OCT, and mERG.
Statistical analysis of the data was carried out using Microsoft Excel on Microsoft Office 2003 and SPSS software (version 16; SPSS Inc., Chicago, Illinois, USA) on Windows 2003.
| Results|| |
Nineteen keratoconic eyes of 19 patients were included in the study. All patients completed 6 months of follow-up and presented with grade II-III keratoconus. Ten patients were women and nine were men. The mean age was 22.00 years (range: 18-35 years).
Visual acuity data is expressed as log MAR ± SD (Snellen value) . [Figure 1] provides the UCVA values for all patients during the preoperative period and at the 6-month follow-up. The mean preoperative UCVA was 0.81 ± 0.10; the mean UCVA at the end of the follow-up period was 0.33 ± 0.1 (P = 0.0001).
The BCVA values preoperatively and at the 6-month follow-up are shown in [Figure 2]. There was a statistically significant (P = 0.0001) improvement in BCVA from the preoperative period to the 6-month follow-up. None of the patients lost lines of BCVA at 6 months compared with the preoperative baseline BCVA.
[Figure 3] shows changes in the spherical equivalent (SEQ) during the follow-up period; at 1 month postoperatively, there was a significant increase in the SEQ followed by a gradual decrease (P = 0.0001). The SEQ reached stabilization at 3 months of follow-up. The mean improvement was 1.5 (±1.0) D. The mean value of the manifest refraction cylinder was used as a measure of the change in the refractive astigmatism. The cylinder values at the 6-month examination were statistically significantly lesser than the preoperative measurements (P = 0.0003). Six months after the cross-linking treatment, the cylinder value decreased by a mean of -1.4 D [Figure 4].
The K-value at the apex (maximum keratometric reading) decreased by a mean of 1.40 D from the preoperative evaluation to 6-month postoperatively (P = 0.0001). [Figure 5] shows the change in the maximum keratometric value at the apex from the preoperative evaluation to 6 months postoperatively. Keratometric changes reached stability at the third month and remained stable until the end of the follow-up period.
Pachymetry measurements (using the Pentacam) at the thinnest location were obtained preoperatively and monthly during the follow-up period until the end of the 6-month follow-up period. At the 1-month postoperative examination, there was a significant reduction in the pachymetry value at the thinnest location (P = 0.0007). The pachymetry value at the thinnest location reduced from 481.16 ± 13.60 mm preoperatively to 458.78 ± 11.28 mm 1-month postoperative. The 3-month evaluation showed the pachymetry values to increase gradually up to 474.94 ± 14.58 mm at the thinnest location, reaching stability at the end of the follow-up period. [Figure 6] shows changes in pachymetry values at the thinnest location over time.
There was a significant increase in the mean IOP of the eyes 1 month after corneal cross-linking (P = 0.0009). The mean preoperative IOP was 12.10 ± 1.62 mmHg, and the mean IOP 1 month after surgery was 14.94 ± 00.91 mmHg. [Figure 7] shows changes in IOP over the follow-up period.
Corneal optical coherence tomography
One month after surgery, four eyes showed remnants of riboflavin in the cornea. At the third month, 13 eyes showed a demarcation line at a depth of 300 ± 10.5 mm, which persisted until the end of the follow-up period.
Endothelial cell count
The endothelial cell count significantly decreased by the end of the follow-up period (P = 0.0009). The preoperative mean endothelial cell count was 2778.63 ± 23.39 cells/mm 2 ; at 6 months of follow-up, the endothelial cell count was 2608.00 ± 208.87 cells/mm 2 . [Figure 8] shows the change in the endothelial cell count during the follow-up period.
Retinal fluorescein angiography
At 6 months of follow-up, fluorescein angiography showed absence of leakage compared with baseline in all studied eyes. No angiographic abnormalities were detected in riboflavin-treated eyes (inflammatory, ischemic, or atrophic).
Retinal optical coherence tomography (foveal central thickness)
The mean FCT at baseline was 229.52 (±19.6). No significant change in the thickness was detected at the end of the follow-up period. In addition, cross-sectional scans of the ETDRS regions showed no changes in riboflavin-treated eyes, without any evidence of pathological changes in the cube scanned. [Figure 9] shows the mean change in FCT during the follow-up period.
Retinal multifocal electroretinography (foveal ring)
mERG results at the foveal ring showed no changes as regards both the P1 amplitude and the implicit time at the end of the follow-up period in riboflavin-treated eyes. [Figure 10]a and b shows the mean mERG changes during the follow-up period. [Figure 11] and [Figure 12] show data from the studied patients.
| Discussion|| |
The key indication for collagen cross-linking is inhibition of the progression of corneal ectasia, such as keratoconus and pellucid marginal degeneration. Collagen cross-linking may also be effective in the treatment and prophylaxis of iatrogenic keratectasia resulting from LASIK. Other than the treatment of keratectasia, the new technique can also be used to treat corneal melting conditions or infectious keratitis because cross-linking strengthens a collagenolytic cornea and UV-A irradiation sterilizes the infectious agent ,.
The increase in biomechanical stiffness after CXL slows the progression of keratoconus and ectasia and, in many cases, improves the patient's visual and topographic outcomes ,,.
In this study, there was an improvement in UCVA and BCVA; none of the patients showed lost lines in BCVA. As regards the SEQ, there was a significant increase in SEQ, followed by a gradual decrease, and the SEQ stabilized at 3 months of follow-up, with a mean improvement of 1.5 (±1.0). This is in accordance with the findings of Coskunseven et al. , who reported an increase in UCVA and BCVA of 0.06 ± 0.05. Coskunseven et al.  reported that the group treated with collagen cross-linking showed a mean decrease in SEQ refraction.
In this study, the maximum K-value decreased by a mean of 1.40 D from the preoperative period to 6 months postoperatively, which was statistically significant. This finding is in concordance with the findings of previous studies ,,,.
As regards the pachymetry measurements at the thinnest location, there was a significant reduction in the pachymetry value at the thinnest location 1 month postoperatively (P = 0.0007). The pachymetry value at the thinnest location reduced from 481.16 ± 13.60 mm preoperatively to 458.78 ± 11.28 mm at 1 month postoperatively. The 3-month evaluation showed pachymetry values to increase gradually to 474.94 ± 14.58 mm at the thinnest location, reaching stability at the end of the follow-up period. Greenstein et al.  reported the same results in their study on pachymetry changes. The physiology of this initial thinning and subsequent rethickening is unclear. Epithelial remodeling is a possible early factor in changes in corneal thickness. Although re-epithelialization after CXL is generally complete 4-5 days after surgery, continued epithelial remodeling could influence the total corneal thickness over time. However, the continued decrease in corneal thickness from 1 to 3 months suggested other causes to be responsible for the changes in corneal thickness, such as compression of collagen fibrils (especially the more transverse-oriented anterior fibrils), changes in corneal hydration, and keratocyte edema ,,.
There was a significant increase in the mean IOP of eyes 1 month after corneal cross-linking (P = 0.0009); this could be explained by increased corneal rigidity and corneal thinning. The findings reported agree with that of an in-vitro study on human corneas, which reported an overestimation of the true IOP measured by Goldmann applanation tonometry .
On anterior segment OCT, four eyes showed remnants of riboflavin in the cornea at 1 month after surgery. At the third month, 13 eyes showed a demarcation line at a depth of 300 ± 10.5 mm, which persisted until the end of the follow-up period. The demarcation line was reported by Caporossi et al. . This also was evidenced by confocal laser scanning microscopy analysis and Visante anterior segment OCT . This high-reflective line (hyperdense) should be interpreted as a pseudoposterior corneal surface ,,, 22. This demarcation line may be responsible for the underestimation of corneal thickness.
In this study the endothelial cell count significantly decreased at the end of the follow-up period (P = 0.0009); however, Caporossi and colleagues reported an insignificant reduction in the endothelial cell count .
As regards posterior segment assessment, retinal fluorescein angiography showed no angiographic abnormalities in riboflavin-treated eyes. The FCT assessed by retinal OCT showed no statistically significant changes.
mERG results at the foveal ring showed no statistically significant changes as regards both the P1 amplitude and the implicit time at the end of the follow-up period in riboflavin-treated eyes. These findings showed the safety of cross-linking in the anatomical and functional elements of the retina.
| Conclusion|| |
CXL is an effective procedure in stopping the progress of keratoconus and is safe for both the anterior and posterior segments.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
|1.||Rabinowitz YS. Keratoconus. Surv Ophthalmol 1998; 42:297-319. |
|2.||Mazzotta C, Balestrazzi A, Traversi C, et al. Treatment of progressive keratoconus by riboflavin-UVA-induced cross-linking of corneal collagen: Uultrastructural analysis by Heidelberg Retinal Tomograph II in vivo confocal microscopy in humans. Cornea 2007; 26:390-397. |
|3.||Coskunseven E, Jankov MR, Hafezi F. Comparative study of corneal collagen cross-linking with Riboflavin and UVA irradiation in patients with keratoconus J Refract Surg 2009; 25:371-376. |
|4.||Vinciguerra P, Albè E, Trazza S, et al. Intraoperative and postoperative effects of corneal collagen cross linking on progressive keratoconus. Arch Ophthalmol 2009; 127:1258-1265. |
|5.||Spadea L. Collagen crosslinking for ectasia following PRK performed in excimer laser-assisted keratoplasty for keratoconus. Eur J Ophthalmol 2012; 22:274-277. |
|6.||Vinciguerra P, Camesasca FI, Albè E, et al. Corneal collagen cross-linking for ectasia after excimer laser refractive surgery: 1-year results. J Refract Surg 2010; 26:486-497. |
|7.||Hersh PS, Greenstein SA, Fry KL. Corneal collagen crosslinking for keratoconus and corneal ectasia: One-year results. J Cataract Refract Surg 2011; 37:149-160. |
|8.||Wollensak G, Aurich H, Wirbelauer C, Sel S. Significance of the riboflavin film in corneal collagen crosslinking. J Cataract Refract Surg 2010; 36:114-120. |
|9.||Hayes S, O′Brart DP, Lamdin LS, Doutch J. Effect of complete epithelial debridement before riboflavin-ultraviolet-A corneal collagen crosslinking therapy. J Cataract Refract Surg 2008; 34:657-661. |
|10.||Filippello M, Stagni E, O′Brart D. Transepithelial corneal collagen crosslinking: Bilateral study. J Cataract Refract Surg2012; 38:283-291. |
|11.||Krumeich JH, Daniel J, Knülle A. Live epikeratophakia for keratoconus. J Cataract Refract Surg 1998; 24:456-463. |
|12.||De Sanctis U, Loiacono C, Richiardi LTurco D, Mutani B, Grignolo FM. Sensitivity and specificity of posterior corneal elevation measured by Pentacam in discriminating keratoconus/subclinical keratoconus. Ophthalmology 2008; 115:1534-1539. |
|13.||Holladay JT. Visual acuity measurements. J Cataract Refract Surg 2004; 30:287-290. |
|14.||De Sanctis u, Missolungi A, Mutani B, Richiardi L, Grignolo FM. Reproducibility and repeatability of central corneal thickness measurement in keratoconus using the rotating Scheimpflug camera and ultrasound pachymetry. Am J Ophthalmol 2007; 144:712-718. |
|15.||Ho JD, Tsai CY, Tsai RJ, et al. Validity of the keratometric index: Evaluation by the Pentacam rotating Scheimpflug camera. J Cataract Refract Surg 2008; 34:137-145. |
|16.||Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003; 135:620-627. |
|17.||Greenstein SA, Shah VP, Fry KL, et al. Corneal thickness changes after corneal collagen crosslinking for keratoconus and corneal ectasia: One-year results. J Cataract Refract Surg 2011; 37:691-700. |
|18.||Caporossi A, Baiocchi S, Mazzotta C, et al. Parasurgical therapy for keratoconus by riboflavin-ultraviolet type A rays induced cross-linking of corneal collagen; preliminary refractive results in an Italian study. J Cataract Refract Surg 2006; 32:837-845. |
|19.||Vinciguerra P, Albè E, Trazza S, et al. Refractive, topographic, tomographic, and aberrometric analysis of keratoconic eyes undergoing corneal cross-linking. Ophthalmology 2009; 116:369-378. |
|20.||Mazzota C,Traversi C, Baiocchi S, et al. Corneal healing after riboflavin ultraviolet-A crosslinking determined by conofocal laser scanning microscopy in vivo: Early and late modifications. Am J Ophthalmol 2008; 146:527-533. |
|21.||Romppainen T, Bachmann LM, Kaufmann C, et al. Effects of riboflavin UVA induced collagen cross linking on intraocular pressure measurement. Invest Ophthalmol Vis Sci 2007; 48 5494-5498. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]